Delwood C. Collins

Long-Term Effect of a First Pregnancy on the Secretion of Prolactin

An early first pregnancy is known to protect against subsequent breast cancer. We speculated that this effect may be mediated by a long-term depression of prolactin secretion after pregnancy. We therefore measured basal and post-stimulation serum levels of prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in two groups--15 women 18 to 23 years of age and 9 women 29 to 40--before and after a first full-term pregnancy, and in 40 appropriate nulliparous controls. We observed no significant change in basal levels of serum LH or FSH or in the levels stimulated by gonadotropin-releasing hormone in any group. A significant decrease was seen, however, in basal and perphenazine-stimulated levels of prolactin after pregnancy in both the younger and older first-pregnancy groups but not in the controls. In a separate cross-sectional study, we compared basal serum prolactin levels in 29 parous and 19 nulliparous women of similar age. The serum prolactin levels were significantly lower in the parous group but were not related to the number of pregnancies (one to three) or the time elapsed (12 to 150 months) since the last delivery. We conclude that a first pregnancy leads to a long-term decrease in serum prolactin secretion, lasting at least 12 to 13 years.

Antiangiogenic and antiproliferative activity of suramin analogues

Abstract The purpose of this study was to test the ability of 70 polyanionic analogues of suramin to inhibit angiogenesis. The ID50, the dose that produced 50% inhibition of angiogenesis, was determined for suramin and each of the analogues by measuring the ability of various amounts to inhibit angiogenesis in vivo in the chick egg chorioallantoic membrane (CAM) assay. Of the 70 analogues, 11 had antiangiogenic activities similar to suramin and an additional 7 were significantly more potent than suramin. All seven of these analogues were from the naphthalenetrisulfonic acid group and contained large urea groups. The benzene sulfonic and disulfonic acid analogues were less active inhibitors of angiogenesis than the naphthalenetrisulfonic acid analogues. Replacement of the naphthalenetrisulfonic acid groups by aliphatic carboxylic acids or benzoic acid gave analogues with very little antiangiogenic activity. In subsequent experiments, the antiproliferative activity of selected analogues on basic FGF (bFGF)-stimulated growth of immortalized human microvascular endothelial cells in vitro was determined. Analogues that inhibited angiogenesis to a greater extent than suramin in the CAM assay generally showed a greater antiproliferative effect on bFGF-induced growth of human microvascular endothelial cells. These results suggest that some of the polyanionic analogues may be potent therapeutic agents for cancers and angiogenesis-dependent diseases.

Sex hormone receptor binding, progestin selectivity, and the new oral contraceptives

The desired biologic effect of progestins used in OCs is progestational activity. Undesired pharmacologic properties such as androgenic activity are not necessary for contraception and increase the potential for adverse effects. A selective progestin has progestational effects at relatively low concentrations or doses and androgenic effects at only relatively high concentrations or doses. The degree to which progestational activity is maximized and androgenic activity is minimized is a measure of a progestins selectivity. The ratio of its affinity for progesterone receptors to its affinity for androgen receptors is the selectivity index. To minimize the androgenic side effects associated with the older progestins, the doses used in OCs have been reduced over the years. These dose reductions have decreased the potential for undesired androgenic effects but also have negatively affected cycle control. Three new progestins, norgestimate, desogestrel, and gestodene, have relatively greater affinity for progesterone receptors than for androgen receptors when compared with the older agents, permitting a reduction in androgenic side effects without the need for further dose reduction. Preclinical receptor-binding studies and animal pharmacologic studies have documented the higher selectivity indexes of these new progestins. Their higher ratios of progestational to androgenic activity provide the basis for the reduction in androgenic adverse effects observed with their clinical use.

Effect of a Peripheral and a Central Acting Opioid Antagonist on the Testicular Response to Stress in Rats

The possible involvement of opioid receptors in mediating the inhibitory effects of immobilization stress on testicular steroidogenesis was determined in adult male rats. Unstressed controls and animals exposed to 3 h of immobilization stress were injected subcutaneously with either vehicle or 1 or 10 mg/kg body weight (BW) of naloxone or naltrexone methobromide (NMB; an opioid receptor antagonist that does not cross the blood-brain barrier) at the beginning of and at 1.5 h of the stress period. Animals were sacrificed at 2 h (30 min after the second injection of antagonist) or 3 h (90 min after the second injection of antagonist) of stress. Plasma LH was not affected by stress, but 30 min after naloxone (1 or 10 mg/kg BW) injection, LH was elevated in both control and stressed rats above levels in vehicle-injected animals. By 90 min after naloxone injection, plasma LH had declined to levels comparable to those in vehicle-injected animals. NMB had no effect on plasma LH concentrations in either control or stressed rats. Three hours of stress reduced plasma testosterone (T) levels by 60% in vehicle-injected animals. This effect of stress on plasma T levels was antagonized by the 10 mg/kg BW dose of naloxone and 1 or 10 mg/kg BW of NMB. The ability of naloxone to reverse the effect of stress on plasma T levels was likely related to its ability to stimulate LH secretion, but NMB normalized plasma T values in stressed animals without altering plasma LH concentrations. Only the highest dose of NMB increased plasma T levels in unstressed control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of diet on lignans and isoflavonoid phytoestrogens in chimpanzees

Diphenolic compounds belonging to the classes of lignans and isoflavonoids have been identified in urine of man and animals, including the chimpanzee. Some of these compounds, formed by intestinal bacteria from plant lignans and phytoestrogens, have been shown in animal studies to exhibit biological activities that suggest they could function as cancer-protective compounds. The effect of diet on urinary excretion of these compounds in the adult male chimpanzee has been studied. It was found that the chimpanzees consuming their regular food excreted large amounts of the isoflavonoid phytoestrogens, equol (mean +/- SE) (127.5 +/- 34.0 nmol/mg cr.) and daidzein (20.7 +/- 9.0 nmol/mg cr.) and the lignan, enterolactone (14.1 + 3.5 nmol/mg cr.). Small amounts of the lignan, enterodiol, (0.4 +/- 0.2 nmol/mg cr.) were also excreted. On all other four test diets (high protein, high carbohydrate, high vegetable, and high fat), the excretion was less, particularly on a high fat diet where the excretion of all diphenolic compounds was reduced by more than 90% to a level observed in omnivorous human subjects or women with breast cancer. These results suggest that diet profoundly influences the excretion of both animal lignans and phytoestrogens in urine. Because non-human primates are particularly resistant to mammary and genital carcinoma on estrogen treatment, the present data suggest that the very high levels of phytoestrogens and lignans as found during exposure to the regular diet may partially account for why these primates are so resistant to hormonal manipulations to induce cancer.

Selectivity information on desogestrel.

Improvement in oral contraceptive formulations was originally achieved through dose reduction of the estrogen and progestogen components. Recently, further improvement was achieved by increasing the selectivity of contraceptive progestins. The ratio between the affinity for the progesterone receptor and the affinity for the androgen receptor is an indicator of progesterone (or androgen) selectivity of a progestin. This ratio (selectivity index) reflects the relative amount of androgenic or progestational effect at a given dose. Relative selectivity can be characterized with in vitro receptor-binding studies and animal pharmacologic experiments. In comparison with levonorgestrel, desogestrel displays markedly lower androgenicity and slightly increased relative progestational activity. In receptor-binding experiments and animal pharmacologic studies, 3-keto-desogestrel, the active metabolite of desogestrel, shows the highest selectivity index. The favorable effect of desogestrel-containing oral contraceptives on lipoprotein metabolism and preexisting androgen-dependent skin disorders and the absence of adverse effects on blood pressure and body weight are attributed to the increased progestin selectivity of desogestrel.

Uptake of suramin by human microvascular endothelial cells

We have demonstrated for the first time that suramin is taken up by human dermal microvascular endothelial (HMEC-1) cells by an active process involving the caveolae system. The uptake of suramin was time-dependent and reduced by more than 90% when incubated in the presence of albumin or at 4 degrees C. Suramin uptake was also inhibited when incubated in the presence of filipin and digitonin, both potent cholesterol-binding agents, but not in the presence of probenecid. The [3H]suramin taken up by the HMEC-1 cells was located primarily within the nucleus, followed by the cytoplasmic fraction. The presence of suramin in these cellular compartments suggests that this drug may act through intracellular mechanisms.

Photoaffinity labeling of rat liver microsomal steroid 5α-reductase by 2-azido-NADP

Abstract Preincubation of female rat liver microsomal preparations with [2′- 32 P]2N 3 -NADP + followed by photolysis with UV lighjt (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of 32 P into at least 3 major protein bands in the molecular weight range of 14–197 Kd. Labeling of a 26 Kd band, the apparent molecular weight of 5α-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP + (100μM) completely inhibited the incorporation of [2′- 32 P]2N 3 -NADP + and preserved the enzyme activity, whereas excess NAD + (100μM) failed to protect 5α-reductase (5αR) activity. Similar results were obtained with the detergent-solubilized form of 5αR. Polyethylene glycol (PEG) fractionation of detergent-solubilized with the detergent-solubilized preparations of 5αR showed that all the 5αR activity could be recovered in the 6.5%, pellet with a 3—4-fold increase in the specific activity. Photolysis of this fraction with [2′- 32 P]2N 3 -NADP + resulted in ∼ 2-fold increase in 32 P labeling of the 5αR band. Increasing photolysis time and concentration of the [2′- 32 P]2N 3 -NADP + indicated that the half-life for photoincorporation and the apparent K d were 1.0 min and 2 μM, respectively. Theser results suggest that 2N 3 -NADP + is an effective probe of the NADP(H) binding site of 5αR, and is a useful marker during purification of the enzyme.

3α-hydroxysteroid dehydrogenase activity in rat liver and skin

Abstract 3α-Hydroxysteroid dehydrogenase (3αHSD) is one of the main enzymes involved in the metabolism of the active androgen, dihydrotestosterone (DHT). 3αHSD catalyzes the reversible reduction of DHT to 5α-androstane-3α, 17β-diol (3αDIOL). The equilibrium of 3αHSD reductive and oxidative activity is an important factor in the regulation of intracellular levels of DHT. In this study, we determined the kinetic characteristics of 3αHSD in the subcellular fractions of female rat liver and abdominal skin. The enzyme expressed its activity in the cytosol and microsomal fractions of both of these tissues. It showed higher activity with the phosphorylated cofactors, NADPH and NADP, and was inhibited by indomethacin. The Vmax values of 3αHSD in the cytosol were 10-fold higher than the Vmax values in the microsomes in both the liver and skin. In both tissues, the Km values with DHT as the substrate (reductive) were lower than the Km with 3αDIOL as the substrate (oxidative). Although the Vmax values of the oxidative reaction were higher than the Vmax values of the reductive reaction in both liver and skin, the low Km values and the higher Vmax/Km ratio for DHT indicated that the reduction of DHT to 3αDIOL was the favored reaction. The enzyme kinetics of 3αHSD suggest that neither tissue accumulates DHT, but promptly converts it to 3αDIOL. When DHT reduction by 3αHSD in the liver and skin was compared on the basis of total tissue weight, the ability to metabolize DHT to 3αDIOL was 12-fold higher in the liver, suggesting that the liver is more effective in DHT reduction to 3αDIOL. (Steroids 59:259–264, 1994)

Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5α-reductase (isozyme-1) I: Analysis of aromatic and hydroxylated amino acid residues

Abstract Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5α-reductase isozyme-1 (r5αR-1) is in a highly conserved region of the polypeptide sequence (residues 160–190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5αR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an ∼40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5αR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an ∼5-fold decrease in Km for NADPH and a significant increase (∼18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5αR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.